Complete Mitochondrial Genome for Lucilia cuprina dorsalis (Diptera: Calliphoridae) from the Northern Territory, Australia.

The Australian sheep blowfly, Lucilia cuprina dorsalis, is a major sheep ectoparasite causing subcutaneous myiasis (flystrike), which can lead to reduced livestock productivity and, in severe instances, death of the affected animals. It is also a primary colonizer of carrion, an efficient pollinator, and used in maggot debridement therapy and forensic investigations. In this study, we report the complete mitochondrial (mt) genome of L. c. dorsalis from the Northern Territory (NT), Australia, where sheep are prohibited animals, unlike the rest of Australia. The mt genome is 15,943 bp in length, comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. The gene order of the current mt genome is consistent with the previously published L. cuprina mt genomes. Nucleotide composition revealed an AT bias, accounting for 77.5% of total mt genome nucleotides. Phylogenetic analyses of 56 species/taxa of dipterans indicated that L. c. dorsalis and L. sericata are the closest among all sibling species of the genus Lucilia, which helps to explain species evolution within the family Luciliinae. This study provides the first complete mt genome sequence for L. c. dorsalis derived from the NT, Australia to facilitate species identification and the examination of the evolutionary history of these blowflies.

First Detection and Circulation of RHDV2 in New Zealand.

Rabbit haemorrhage disease virus 2 (RHDV2) is a highly pathogenic lagovirus that causes lethal disease in rabbits and hares (lagomorphs). Since its first detection in Europe in 2010, RHDV2 has spread worldwide and has been detected in over 35 countries so far. Here, we provide the first detailed report of the detection and subsequent circulation of RHDV2 in New Zealand. RHDV2 was first detected in New Zealand in 2018, with positive samples retrospectively identified in December 2017. Subsequent time-resolved phylogenetic analysis suggested a single introduction into the North Island between March and November 2016. Genetic analysis identified a GI.3P-GI.2 variant supporting a non-Australian origin for the incursion; however, more accurate identification of the source of the incursion remains challenging due to the wide global distribution of the GI.3P-GI.2 variant. Furthermore, our analysis suggests the spread of the virus between the North and South Islands of New Zealand at least twice, dated to mid-2017 and around 2018. Further phylogenetic analysis also revealed a strong phylogeographic pattern. So far, no recombination events with endemic benign New Zealand rabbit caliciviruses have been identified. This study highlights the need for further research and surveillance to monitor the distribution and diversity of lagoviruses in New Zealand and to detect incursions of novel variants.

Utilizing Molecular Epidemiology and Citizen Science for the Surveillance of Lagoviruses in Australia.

Australia has multiple lagoviruses with differing pathogenicity. The circulation of these viruses was traditionally determined through opportunistic sampling events. In the lead up to the nationwide release of RHDVa-K5 (GI.1aP-GI.1a) in 2017, an existing citizen science program, RabbitScan, was augmented to allow members of the public to submit samples collected from dead leporids for lagovirus testing. This study describes the information obtained from the increased number of leporid samples received between 2015 and 2022 and focuses on the recent epidemiological interactions and evolutionary trajectory of circulating lagoviruses in Australia between October 2020 and December 2022. A total of 2771 samples were tested from January 2015 to December 2022, of which 1643 were lagovirus-positive. Notable changes in the distribution of lagovirus variants were observed, predominantly in Western Australia, where RHDV2-4c (GI.4cP-GI.2) was detected again in 2021 after initially being reported to be present in 2018. Interestingly, we found evidence that the deliberately released RHDVa-K5 was able to establish and circulate in wild rabbit populations in WA. Overall, the incorporation of citizen science approaches proved to be a cost-efficient method to increase the sampling area and enable an in-depth analysis of lagovirus distribution, genetic diversity, and interactions. The maintenance of such programs is essential to enable continued investigations of the critical parameters affecting the biocontrol of feral rabbit populations in Australia, as well as to enable the detection of any potential future incursions.

A quantitative risk assessment for the incursion of lumpy skin disease virus into Australia via long-distance windborne dispersal of arthropod vectors.

Lumpy skin disease (LSD) is an infectious disease of cattle and water buffalo caused by lumpy skin disease virus (LSDV). It is primarily transmitted mechanically by biting insects. LSDV has spread from Africa to the Middle-East, the Balkans, Caucasus, Russia, Kazakhstan, China, Asia and India, suggesting that a wide variety of arthropod vectors are capable of mechanical transmission. In 2022, LSD was detected in Indonesia, heightening awareness for Australia's livestock industries. To better understand the risk of LSDV incursion to Australia we undertook a quantitative risk assessment (QRA) looking at windborne dispersal of arthropod vectors, assuming a hypothetical situation where LSD is endemic in south-east Asia and Papua New Guinea. We estimated the risk of LSDV incursion to be low, with a median incursion rate of one incursion every 403 years, based on a model where several infectious insects (i.e. a 'small batch' of 3-5) must bite a single bovine to transmit infection. The incursion risk increases substantially to one incursion every 7-8 years if a bite from a single insect is sufficient for transmission. The risk becomes negligible (one incursion every 20,706 years) if bites from many insects (i.e. a 'large batch' of 30-50 insects) are necessary. Critically, several of our parameter estimates were highly uncertain during sensitivity analyses. Thus, a key outcome of this QRA was to better prioritise surveillance activities and to understand the key research gaps associated with LSDV in the Australasian context. The current literature shows that multiple vectors are required for successful bovine-to-vector transmission of LSDV, suggesting that our estimate of one outbreak every 403 years more accurately represents the risk to Australia; however, the role of single insects in transmission has not yet been evaluated. Similarly, attempts to transmit LSDV between bovines by Culicoides have not been successful, although midges were the highest risk vector category in our model due to the high vector-to-host ratio for midges compared to other vector categories. Our findings provide further insight into the risk of LSD to Australian cattle industries and identify the Tiwi Islands and areas east of Darwin as priority regions for LSDV surveillance, especially between December and March.

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses.

The genus Lagovirus of the family Caliciviridae contains some of the most virulent vertebrate viruses known. Lagoviruses infect leporids, such as rabbits, hares and cottontails. Highly pathogenic viruses such as Rabbit haemorrhagic disease virus 1 (RHDV1) cause a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 h of infection, killing over 95 % of susceptible animals. Research into the pathophysiological mechanisms that are responsible for this extreme phenotype has been hampered by the lack of a reliable culture system. Here, we report on a new ex vivo model for the cultivation of lagoviruses in cells derived from the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus). We show that three different lagoviruses, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids, but not in monolayer cultures derived from cat (Felis catus) or mouse (Mus musculus) organoids. Virus multiplication was demonstrated by (i) an increase in viral RNA levels, (ii) the accumulation of dsRNA viral replication intermediates and (iii) the expression of viral structural and non-structural proteins. The establishment of an organoid culture system for lagoviruses will facilitate studies with considerable implications for the conservation of endangered leporid species in Europe and North America, and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

Sustained Impact of RHDV2 on Wild Rabbit Populations across Australia Eight Years after Its Initial Detection.

Following the arrival of rabbit haemorrhagic disease virus 2 (RHDV2) in Australia, average rabbit population abundances were reduced by 60% between 2014 and 2018 based on monitoring data acquired from 18 sites across Australia. During this period, as the seropositivity to RHDV2 increased, concurrent decreases were observed in the seroprevalence of both the previously circulating RHDV1 and RCVA, a benign endemic rabbit calicivirus. However, the detection of substantial RHDV1 seropositivity in juvenile rabbits suggested that infections were continuing to occur, ruling out the rapid extinction of this variant. Here we investigate whether the co-circulation of two pathogenic RHDV variants was sustained after 2018 and whether the initially observed impact on rabbit abundance was still maintained. We monitored rabbit abundance and seropositivity to RHDV2, RHDV1 and RCVA at six of the initial eighteen sites until the summer of 2022. We observed sustained suppression of rabbit abundance at five of the six sites, with the average population reduction across all six sites being 64%. Across all sites, average RHDV2 seroprevalence remained high, reaching 60-70% in adult rabbits and 30-40% in juvenile rabbits. In contrast, average RHDV1 seroprevalence declined to <3% in adult rabbits and 5-6% in juvenile rabbits. Although seropositivity continued to be detected in a low number of juvenile rabbits, it is unlikely that RHDV1 strains now play a major role in the regulation of rabbit abundance. In contrast, RCVA seropositivity appears to be reaching an equilibrium with that of RHDV2, with RCVA seroprevalence in the preceding quarter having a strong negative effect on RHDV2 seroprevalence and vice versa, suggesting ongoing co-circulation of these variants. These findings highlight the complex interactions between different calicivirus variants in free-living rabbit populations and demonstrate the changes in interactions over the course of the RHDV2 epizootic as it has moved towards endemicity. While it is encouraging from an Australian perspective to see sustained suppression of rabbit populations in the eight years following the arrival of RHDV2, it is likely that rabbit populations will eventually recover, as has been observed with previous rabbit pathogens.

Rabbit haemorrhagic disease virus 2 from Singapore 2020 outbreak revealed an Australian recombinant variant.

Rabbit haemorrhagic disease (RHD) is a significant and debilitating viral disease affecting lagomorphs. In September 2020, Singapore reported its first cases of RHD virus (RHDV) infection in domesticated rabbits. The initial findings reported that the outbreak strain belonged to genotype GI.2 (RHDV2/RHDVb), and epidemiological investigations could not identify the definitive source of the virus origin. Further recombination detection and phylogenetic analyses of the Singapore outbreak strain revealed that the RHDV was a GI.2 structural (S)/GI.4 non-structural (NS) recombinant variant. Sequence analyses on the National Centre for Biotechnology Information (NCBI) database showed high homology to recently emerged Australian variants, which were prevalent in local Australian lagomorph populations since 2017. Time-structured and phylogeographic analyses for the S and NS genes revealed a close genetic relationship between the Singapore RHDV strain and the Australian RHDV variants. More thorough epidemiological inquiries are necessary to ascertain how an Australian RHDV was introduced into the Singapore rabbit population, and opportune development of RHDV diagnostics and vaccines will be important to safeguard lagomorphs from future RHDV infection and disease management.

Public health interventions successfully mitigated multiple incursions of SARS-CoV-2 Delta variant in the Australian Capital Territory.

The COVID-19 pandemic has presented a unique opportunity to understand how real-time pathogen genomics can be used for large-scale outbreak investigations. On 12 August 2021, the Australian Capital Territory (ACT) detected an incursion of the SARS-CoV-2 Delta (B.1.617.2) variant. Prior to this date, SARS-CoV-2 had been eliminated locally since 7 July 2020. Several public health interventions were rapidly implemented in response to the incursion, including a territory-wide lockdown and comprehensive contact tracing. The ACT has not previously used pathogen genomics at a population level in an outbreak response; therefore, this incursion also presented an opportunity to investigate the utility of genomic sequencing to support contact tracing efforts in the ACT. Sequencing of >75% of the 1793 laboratory-confirmed cases during the 3 months following the initial notification identified at least 13 independent incursions with onwards spread in the community. Stratification of cases by genomic cluster revealed that distinct cohorts were affected by the different incursions. Two incursions resulted in most of the community transmission during the study period, with persistent transmission in vulnerable sections of the community. Ultimately, both major incursions were successfully mitigated through public health interventions, including COVID-19 vaccines. The high rates of SARS-CoV-2 sequencing in the ACT and the relatively small population size facilitated detailed investigations of the patterns of virus transmission, revealing insights beyond those gathered from traditional contact tracing alone. Genomic sequencing was critical to disentangling complex transmission chains to target interventions appropriately.

Does 2x2 airplane passenger contact tracing for infectious respiratory pathogens work? A systematic review of the evidence.

We critically appraised the literature regarding in-flight transmission of a range of respiratory infections to provide an evidence base for public health policies for contact tracing passengers, given the limited pathogen-specific data for SARS-CoV-2 currently available. Using PubMed, Web of Science, and other databases including preprints, we systematically reviewed evidence of in-flight transmission of infectious respiratory illnesses. A meta-analysis was conducted where total numbers of persons on board a specific flight was known, to calculate a pooled Attack Rate (AR) for a range of pathogens. The quality of the evidence provided was assessed using a bias assessment tool developed for in-flight transmission investigations of influenza which was modelled on the PRISMA statement and the Newcastle-Ottawa scale. We identified 103 publications detailing 165 flight investigations. Overall, 43.7% (72/165) of investigations provided evidence for in-flight transmission. H1N1 influenza A virus had the highest reported pooled attack rate per 100 persons (AR = 1.17), followed by SARS-CoV-2 (AR = 0.54) and SARS-CoV (AR = 0.32), Mycobacterium tuberculosis (TB, AR = 0.25), and measles virus (AR = 0.09). There was high heterogeneity in estimates between studies, except for TB. Of the 72 investigations that provided evidence for in-flight transmission, 27 investigations were assessed as having a high level of evidence, 23 as medium, and 22 as low. One third of the investigations that reported on proximity of cases showed transmission occurring beyond the 2x2 seating area. We suggest that for emerging pathogens, in the absence of pathogen-specific evidence, the 2x2 system should not be used for contact tracing. Instead, alternate contact tracing protocols and close contact definitions for enclosed areas, such as the same cabin on an aircraft or other forms of transport, should be considered as part of a whole of journey approach.

Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization.

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they 'chaperone' p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes.

Pathogen profiling of Australian rabbits by metatranscriptomic sequencing.

Australia is known for its long history of using biocontrol agents, such as myxoma virus (MYXV) and rabbit haemorrhagic disease virus (RHDV), to manage wild European rabbit populations. Interestingly, while undertaking RHDV surveillance of rabbits that were found dead, we observed that approximately 40% of samples were negative for RHDV. To investigate whether other infectious agents are responsible for killing rabbits in Australia, we subjected a subset of these RHDV-negative liver samples to metatranscriptomic sequencing. In addition, we investigated whether the host transcriptome data could provide additional differentiation between likely infectious versus non-infectious causes of death. We identified transcripts from several Clostridia species, Pasteurella multocida, Pseudomonas spp., and Eimeria stiedae, in liver samples of several rabbits that had died suddenly, all of which are known to infect rabbits and are capable of causing disease and mortality. In addition, we identified Hepatitis E virus and Cyniclomyces yeast in some samples, both of which are not usually associated with severe disease. In one-third of the sequenced total liver RNAs, no infectious agent could be identified. While metatranscriptomic sequencing cannot provide definitive evidence of causation, additional host transcriptome analysis provided further insights to distinguish between pathogenic microbes and commensals or environmental contaminants. Interestingly, three samples where no pathogen could be identified showed evidence of up-regulated host immune responses, while immune response pathways were not up-regulated when E. stiedae, Pseudomonas, or yeast were detected. In summary, although no new putative rabbit pathogens were identified, this study provides a robust workflow for future investigations into rabbit mortality events.

Immunological Cross-Protection between Different Rabbit Hemorrhagic Disease Viruses-Implications for Rabbit Biocontrol and Vaccine Development.

The use of rabbit hemorrhagic disease virus (RHDV) as a biocontrol agent to control feral rabbit populations in Australia, in combination with circulating endemic strains, provides a unique environment to observe the interactions between different lagoviruses competing for the same host. Following the arrival of RHDV2 (GI.2) in Australia, it became necessary to investigate the potential for immunological cross-protection between different variants, and the implications of this for biocontrol programs and vaccine development. Laboratory rabbits of various immune status-(1) rabbits with no detectable immunity against RHDV; (2) rabbits with experimentally acquired immunity after laboratory challenge; (3) rabbits immunised with a GI.2-specific or a multivalent RHDV inactivated virus prototype vaccine; or (4) rabbits with naturally acquired immunity-were challenged with one of three different RHDV variants (GI.1c, GI.1a or GI.2). The degree of cross-protection observed in immune rabbits was associated with the variant used for challenge, infectious dose of the virus and age, or time since acquisition of the immunity, at challenge. The immune status of feral rabbit populations should be determined prior to intentional RHDV release because of the high survival proportions in rabbits with pre-existing immunity. In addition, to protect domestic rabbits in Australia, a multivalent RHDV vaccine should be considered because of the limited cross-protection observed in rabbits given monovalent vaccines.

Cross-protection, infection and case fatality rates in wild European rabbits experimentally challenged with different rabbit haemorrhagic disease viruses.

Rabbit haemorrhagic disease virus 2 (RHDV2) is now the dominant calicivirus circulating in wild rabbit populations in Australia. This study compared the infection and case fatality rates of RHDV2 and two RHDVs in wild rabbits, as well as their ability to overcome immunity to the respective other strains. Wild rabbits were allocated to groups either blindly or based on pre-screening for RHDV/RHDV2 antibodies at capture. Rabbits were monitored regularly until their death or humane killing at 7 days post infection. Liver and eyeball samples were collected for lagovirus testing and aging rabbits, respectively. At capture, rabbits showed high seroprevalence to RHDV2 but not to RHDV. In RHDV/RHDV2 seronegative rabbits at capture, infection rates were highest in those inoculated with RHDV2 (81.8%, 18 out of 22), followed by K5 (53.8%, seven out of 13) and CZECH (40.0%, two out of five), but these differences were not statistically significant. In rabbits with previous exposure to RHDV2 at capture, infection rates were highest when inoculated with K5 (59.6%, 31 out of 52) followed by CZECH (46.0%, 23 out of 50), with infection rates higher in younger rabbits for both viruses. In RHDV/RHDV2 seronegative rabbits at capture, case fatality rates were highest for those inoculated with K5 (71.4%), followed by RHDV2 (50.0%) and CZECH (50.0%). In rabbits with previous exposure to RHDV2 at capture, case fatality rates were highest in rabbits inoculated with K5 (12.9%) followed by CZECH (8.7%), with no case fatalities following RHDV2 inoculation. Case fatality rates did not differ significantly between inoculums in either serostatus group at capture. Based on multivariable modelling, time to death post RHDV inoculation increased in rabbits with recent RHDV2 exposure compared with seronegative rabbits and with age. The results suggest that RHDV2 may cause higher mortalities than other variants in seronegative rabbit populations but that K5 may be more effective in reducing rabbit populations in an RHDV2-dominant landscape.

Changes in virus transmission dynamics following the emergence of RHDV2 shed light on its competitive advantage over previously circulating variants.

Rabbit haemorrhagic disease virus (RHDV) is highly pathogenic to European rabbits. Until recently, only one serotype of RHDV was known, GI.1/RHDV. RHDV2/GI.2 is a novel virus that has rapidly spread and become the dominant pathogenic calicivirus in wild rabbits worldwide. It is speculated that RHDV2 has three competitive advantages over RHDV: (a) the ability to partially overcome immunity to other variants; (b) the ability to clinically infect young rabbits; and (c) a wider host range. These differences would be expected to influence virus transmission dynamics. We used markers of recent infection (IgM/IgA antibodies) to investigate virus transmission dynamics pre and post the arrival of RHDV2. Our data set contained over 3,900 rabbits sampled across a 7-year period at 12 Australian sites. Following the arrival of RHDV2, seasonal peaks in IgM and IgA seropositivity shifted forward one season, from winter to autumn and spring to winter, respectively. Contrary to predictions, we found only weak effects of rabbit age, seropositivity to non-pathogenic calicivirus RCV-A1 and population abundance on IgM/IgA seropositivity. Our results demonstrate that RHDV2 enters rabbit populations shortly after the commencement of annual breeding cycles. Upon entering, the population RHDV2 undergoes extensive replication in young rabbits, causing clinical disease, high virus shedding, mortality and the creation of virus-laden carcasses. This results in high virus contamination in the environment, furthering the transmission of RHDV2 and initiating outbreaks, whilst simultaneously removing the susceptible cohort required for the effective transmission of RHDV. Although RHDV may enter the population at the same time point, it is sub-clinical in young rabbits, causing minimal virus shedding and low environmental contamination. Our results demonstrate a major shift in epidemiological patterns in virus transmission, providing the first evidence that RHDV2's ability to clinically infect young rabbits is a key competitive advantage in the field.

Comparative Epidemiology of Rabbit Haemorrhagic Disease Virus Strains from Viral Sequence Data.

Since their introduction in 1859, European rabbits (Oryctolagus cuniculus) have had a devastating impact on agricultural production and biodiversity in Australia, with competition and land degradation by rabbits being one of the key threats to agricultural and biodiversity values in Australia. Biocontrol agents, with the most important being the rabbit haemorrhagic disease virus 1 (RHDV1), constitute the most important landscape-scale control strategies for rabbits in Australia. Monitoring field strain dynamics is complex and labour-intensive. Here, using phylodynamic models to analyse the available RHDV molecular data, we aimed to: investigate the epidemiology of various strains, use molecular data to date the emergence of new variants and evaluate whether different strains are outcompeting one another. We determined that the two main pathogenic lagoviruses variants in Australia (RHDV1 and RHDV2) have had similar dynamics since their release, although over different timeframes (substantially shorter for RHDV2). We also found a strong geographic difference in their activities and evidence of overall competition between the two viruses.

Distribution and Genetic Diversity of Hepatitis E Virus in Wild and Domestic Rabbits in Australia.

In 2020, Hepatitis E virus (HEV) was detected for the first time in Australian rabbits. To improve our understanding of the genetic diversity and distribution of the virus, 1635 rabbit liver samples from locations across Australia were screened via RT-qPCR for HEV. HEV genomes were amplified and sequenced from 48 positive samples. Furthermore, we tested 380 serum samples from 11 locations across Australia for antibodies against HEV. HEV was detected in rabbits from all states and territories, except the Northern Territory. Seroprevalence varied between locations (from 0% to 22%), demonstrating that HEV is widely distributed in rabbit populations across Australia. Phylogenetic analyses showed that Australian HEV sequences are genetically diverse and that HEV was likely introduced into Australia independently on several occasions. In summary, this study broadens our understanding of the genetic diversity of rabbit HEV globally and shows that the virus is endemic in both domestic and wild rabbit populations in Australia.

Frequent intergenotypic recombination between the non-structural and structural genes is a major driver of epidemiological fitness in caliciviruses.

The diversity of lagoviruses (Caliciviridae) in Australia has increased considerably in recent years. By the end of 2017, five variants from three viral genotypes were present in populations of Australian rabbits, while prior to 2014 only two variants were known. To understand the evolutionary interactions among these lagovirus variants, we monitored their geographical distribution and relative incidence over time in a continental-scale competition study. Within 3 years of the incursion of rabbit haemorrhagic disease virus 2 (RHDV2, denoted genotype GI.1bP-GI.2 [polymerase genotype]P-[capsid genotype]) into Australia, two novel recombinant lagovirus variants emerged: RHDV2-4e (genotype GI.4eP-GI.2) in New South Wales and RHDV2-4c (genotype GI.4cP-GI.2) in Victoria. Although both novel recombinants contain non-structural genes related to those from benign, rabbit-specific, enterotropic viruses, these variants were recovered from the livers of both rabbits and hares that had died acutely. This suggests that the determinants of host and tissue tropism for lagoviruses are associated with the structural genes, and that tropism is intricately connected with pathogenicity. Phylogenetic analyses demonstrated that the RHDV2-4c recombinant emerged independently on multiple occasions, with five distinct lineages observed. Both the new RHDV2-4e and -4c recombinant variants replaced the previous dominant parental RHDV2 (genotype GI.1bP-GI.2) in their respective geographical areas, despite sharing an identical or near-identical (i.e. single amino acid change) VP60 major capsid protein with the parental virus. This suggests that the observed replacement by these recombinants was not driven by antigenic variation in VP60, implicating the non-structural genes as key drivers of epidemiological fitness. Molecular clock estimates place the RHDV2-4e recombination event in early to mid-2015, while the five RHDV2-4c recombination events occurred from late 2015 through to early 2017. The emergence of at least six viable recombinant variants within a 2-year period highlights the high frequency of these events, detectable only through intensive surveillance, and demonstrates the importance of recombination in lagovirus evolution.

Passive Immunisation against RHDV2 Induces Protection against Disease but Not Infection.

Rabbit haemorrhagic disease virus 2 (RHDV2) is a lagovirus in the family Caliciviridae. The closely related Rabbit haemorrhagic disease virus (RHDV, termed RHDV1 throughout this manuscript for clarity) has been used extensively as a biocontrol agent in Australia since the mid-1990s to manage wild rabbit populations, a major economic and environmental pest species. Releasing RHDV1 into populations with a high proportion of rabbits less than 8-10 weeks of age leads to non-lethal infection in many of these young animals, with subsequent seroconversion and long-term immunity against reinfection. In contrast, RHDV2 causes lethal disease even in young rabbits, potentially offering substantial benefits for rabbit management programs over RHDV1. However, it is not clear how acquired resistance from maternal antibodies may influence immunity after RHDV2 infection. In this study, we assessed serological responses after RHDV2 challenge in young rabbits of three different ages (5-, 7-, or 9-weeks-old) that were passively immunised with either high- (titre of 2560 by RHDV IgG ELISA; 2.41 mg/mL total protein) or low- (titre of 160-640 by RHDV IgG ELISA; 1.41 mg/mL total protein) dose RHDV2 IgG to simulate maternal antibodies. All rabbits treated with a high dose and 75% of those treated with a low dose of RHDV2 IgG survived virus challenge. Surviving animals developed robust lagovirus-specific IgA, IgM, and IgG responses within 10 days post infection. These findings demonstrate that the protection against RHDV2 conferred by passive immunisation is not sterilising. Correspondingly, this suggests that the presence of maternal antibodies in wild rabbit populations may impede the effectiveness of RHDV2 as a biocontrol.

Calicivirus Non-structural Proteins: Potential Functions in Replication and Host Cell Manipulation.

The Caliciviridae are a family of viruses with a single-stranded, non-segmented RNA genome of positive polarity. The ongoing discovery of caliciviruses has increased the number of genera in this family to 11 (Norovirus, Nebovirus, Sapovirus, Lagovirus, Vesivirus, Nacovirus, Bavovirus, Recovirus, Salovirus, Minovirus, and Valovirus). Caliciviruses infect a wide range of hosts that include fishes, amphibians, reptiles, birds, and marine and land mammals. All caliciviruses have a genome that encodes a major and a minor capsid protein, a genome-linked viral protein, and several non-structural proteins. Of these non-structural proteins, only the helicase, protease, and RNA-dependent RNA polymerase share clear sequence and structural similarities with proteins from other virus families. In addition, all caliciviruses express two or three non-structural proteins for which functions have not been clearly defined. The sequence diversity of these non-structural proteins and a multitude of processing strategies suggest that at least some have evolved independently, possibly to counteract innate and adaptive immune responses in a host-specific manner. Studying these proteins is often difficult as many caliciviruses cannot be grown in cell culture. Nevertheless, the study of recombinant proteins has revealed many of their properties, such as intracellular localization, capacity to oligomerize, and ability to interact with viral and/or cellular proteins; the release of non-structural proteins from transfected cells has also been investigated. Here, we will summarize these findings and discuss recent in silico studies that identified previously overlooked putative functional domains and structural features, including transmembrane domains that suggest the presence of viroporins.

Age and Infectious Dose Significantly Affect Disease Progression after RHDV2 Infection in Naïve Domestic Rabbits.

Rabbit haemorrhagic disease virus 2 (RHDV2 or GI.2, referring to any virus with lagovirus GI.2 structural genes) is a recently emerged calicivirus that causes generalised hepatic necrosis and disseminated intravascular coagulation leading to death in susceptible lagomorphs (rabbits and hares). Previous studies investigating the virulence of RHDV2 have reported conflicting results, with case fatality rates ranging from 0% to 100% even within a single study. Lagoviruses are of particular importance in Australia and New Zealand where they are used as biocontrol agents to manage wild rabbit populations, which threaten over 300 native species and result in economic impacts in excess of $200 million AUD annually to Australian agricultural industries. It is critically important that any pest control method is both highly effective (i.e., virulent, in the context of viral biocontrols) and has minimal animal welfare impacts. To determine whether RHDV2 might be a suitable candidate biocontrol agent, we investigated the virulence and disease progression of a naturally occurring Australian recombinant RHDV2 in both 5-week-old and 11-week-old New Zealand White laboratory rabbits after either high or low dose oral infection. Objective measures of disease progression were recorded through continuous body temperature monitoring collars, continuous activity monitors, and twice daily observations. We observed a 100% case fatality rate in both infected kittens and adult rabbits after either high dose or low dose infection. Clinical signs of disease, such as pyrexia, weight loss, and reduced activity, were evident in the late stages of infection. Clinical disease, i.e., welfare impacts, were limited to the period after the onset of pyrexia, lasting on average 12 h and increasing in severity as disease progressed. These findings confirm the high virulence of this RHDV2 variant in naïve rabbits. While age and infectious dose significantly affected disease progression, the case fatality rate was consistently 100% under all conditions tested.